1. Field of the Invention
This invention relates to a method for producing recombinant viruses and vectors for the same. Furthermore, this invention relates to deoxyribonucleic acid (DNA) constructs for the method and the recombinant viruses produced by the method.
2. Description of the Related Art
A recombinant viral vector means a genetically engineered vector, which is used in, for example: the preparation of a vaccine; analysis of function of a gene, a group of genes, or a genomic domain; production of proteins; and gene therapy. Genomic materials, which can be integrated into a viral vector, include any genomic materials such as a gene, cDNA, genomic DNA, DNA sequences encoding peptides or proteins, ribonucleic acid (RNA), anti-sense RNA, siRNA, DNA for si RNA, a promoter, or an enhancer. A virus, which is used in the preparation of a vector, includes baculovirus, adenovirus, adeno-associated virus (AAV), retrovirus, Herpes virus, hepatitis B virus (HBV), polioma virus, sindbid virus, and vaccinia virus.
Retroviruses are known as one of the most widely used viruses in gene therapy. See Patience, C., et al., Packaging of Endogenous Retroviral Sequences in Retroviral Vectors Produced by Murine and Human Packaging Cells, J. Virol, April 1998, pp. 2671-2676, Vol. 72, No. 4; Marshall, E., Gene Therapy's Growing Pains, Science, Aug. 25, 1995, pp. 1050-1055, Vol. 269. Retroviral genomes have two long terminal repeats (LTRs), capsid sequences and 3 (three) coding regions (gag, pol and env), and the method of preparation and their use in vitro and in vivo have been disclosed. See WO9908692A1 and EP 453242. The clinical application of a retroviral vector, however, has the following disadvantages: it forms a replication-competent retrovirus (RCR) (see Powell, S. K., et al., Efficacy of Antiretroviral Agents Against Murine Replication-Competent Retrovirus Infection in Human Cells, J. Virol., October 1999, pp. 8813-8816, Vol. 73, No. 10); it has a relatively lower level of gene expression, and the level decreases continuously in vivo; and the titer of virus is low. Furthermore, the retrovirus cannot transfer a gene to a non-dividing cell. See Jaffee, E. M., et al., High Efficiency Gene Transfer into Primary Human Tumor Explants Without Cell Selection, Cancer Research, 1993, pp. 2221-2226, Vol. 53, Issue 10; Bender, M. A., et al., Evidence that the Packaging Signal of Moloney Murine Leukemia Virus Extends into the gag Region, J. Virol., May 1987, pp. 1639-1646, Vol. 61, No. 5.
An adenovirus has a linear genome (sized about 36 kb) and includes replication origin and capsid signals near to the left inverse terminal region (ITR) (103 bp). See Shenk, T., Adenoviridae: The Viruses and Their Replication, Fields Virology, 3rd Ed., 1996, pp. 2111-2148. Usually, adenoviral vectors have been made by deleting the E1 site of the viral genome, which is essential for viral replication. The vector includes foreign genomic material substituting the E1 site, which are transferred to packaging cells which provide E1 proteins. As a result, viral replication occurs only in the packaging cells. Representative packaging cells include, for example, HEK 293 cell, 911 cell, and PER.C6 cells. See Hitt, M. M., et al., Human Adenovirus Vectors for Gene Transfer into Mammalian Cells, Advances in Pharmacology, 1997, pp. 137-206, Vol. 40; Wang, Y., et al., Adenovirus Technology for Gene Manipulation and Functional Studies, Drug Discovery Today, January 2000, pp. 10-16, Vol. 5, No. 1. An adenoviral vector has advantages such as safety, affinity to various cells, possibility of infecting dividing cells and higher titer (1011 pfu/ml). Thus, adenoviruses have been used in the preparation of a vector expressing heterologous genes.
Adeno-associated virus (sized about 4700 bp) has ITRs (sized about 145 bp) as replication origins at each terminus. It can be integrated into the genomic DNA of various types of host cells safely and specifically.
In order to use a recombinant virus as a vector, it should be modified not to replicate in the infected cells. Therefore, so-called “defective viruses”, which have deletions of some essential regions of genome for viral replication, are usually employed in the preparation of recombinant viral vectors. In a retrovirus, for example, gag, pol and/or env genes are deleted, and the regions are replaced with desired genomic materials. See Bender, M. A., et al., Evidence that the Packaging Signal of Moloney Murine Leukemia Virus Extends into the gag Region, J. Virol., May 1987, pp. 1639-1646, Vol. 61, No. 5. For an adenovirus, the E1, E2 and/or E4 sites are deleted for the preparation of a viral vector. See Levrero, M., et al., Defective and Nondefective Adenovirus Vectors for Expressing Foreign Genes It Vitro and In Vivo, Gene, 1991, pp. 195-202, Vol. 101; Ghosh-Choudhury, G., et al., Human Adenovirus Cloning Vectors Based on Infectious Bacterial Plasmids, Gene, 1986, pp. 161-171, Vol. 50. Additionally, a pseudo-virus vector, which consists of only the essential regions (such as, ITR and capsid sequences) of the replication, has also been used for obtaining recombinant viruses. See WO95/02697.
In addition, baculoviruses, which are known as having circular genomic DNA, have been used for the preparation of viral vectors. Baculoviridae are infectious in invertebrates, such as insects and crustacea. Autographa californica nuclear polyhedrosis virus (AcNPV) is one of the most widely used baculoviruses, and it contains double-stranded circular genomic DNA (sized about 134 kb) (GenBank: NC—001623). Smith et al. developed recombinant baculoviruses by inserting β-interferon genes, and successfully obtained interferon proteins from insect cells using the recombinant baculoviruses. See Smith, G. E., et al., Production of Human Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector, Molecular and Cellular Biology, December 1983, pp. 2156-2165, Vol. 3, No. 12. Since then, numerous genes have been expressed and produced using a recombinant baculoviral system. The features of the methods are that: i) it is possible to produce desired recombinant baculoviruses at high efficiency by employing polyhedrin promoter; and ii) it can be used for the expression of genes that do not exhibit their activities when incubated in bacteria. This is due to that post-translational modification is carried out in insect cells. However, the size of the genome of baculovirus made it difficult to engineer the genome through the conventional restriction and ligation method. Therefore, homologous recombination has been used in insect cells generally. In order to conduct homologous recombination, carrier vectors containing desired genes and nucleic acid sequences necessary for the homologous recombination are prepared. Then, insect cells are transfected with the carrier vectors and viral genomic DNA to induce recombination. In that method, however, since the production efficiency of recombinant viruses is relatively low (0.1-2%), repetitive screening of baculoviral plaques should be performed in order to obtain the desired recombinant viruses. Thus, it is also considered as being an inefficient method.
Kitts et al. disclosed an improved method that increased production efficiency of recombinant viruses. See Kitts, P. A., Linearization of Baculovirus DNA Enhances the Recovery of Recombinant Virus Expression Vectors, Nucleic Acids Research, 1990, pp. 5667-5672, Vol. 18, No. 19. They made baculoviral genomic DNA to be in linear form using a restriction enzyme. In the Kitts' method, a linker, which includes Bsu36I recognition base sequences, CCTNAGG, or lacZ genes, is inserted into wild-type AcNPV genome at the polyherin locus in order to introduce Bsu36I site recognition nucleic acid sequences. Next, the viral DNA is digested with Bsu36I restriction enzyme, and then transferred to insect cells together with carrier vectors containing desired genomic materials so as to induce homologous recombination. The production efficiency of the desired recombinant viruses is from 26% to 44%. According to that method, linear digested DNA segments, which are not subjected to recombination, are excluded selectively from the production of viral vectors. Thus, the desired recombinant viruses expressing desired protein can be generated with relatively high efficiency. However, the method has disadvantages as follows: i) it is not possible to digest Bsu36I recognition sites up to 100% and ii) since the digested DNA segments are fused with each other in insect cells by ligase, selection procedures for the desired viral vectors need to be employed.
Peakman et al. provided another method using site-specific recombination of cre/loxP system in vitro. See Peakman, T. C., et al., Highly Efficient Generation of recombinant Baculoviruses by Enzymatically Mediated Site-Specific In vitro Recombination, Nucleic Acids Research, pp. 495-500, Vol. 20, No. 3. Briefly, they prepared baculoviruses containing loxP sites and applied them to site-specific recombination in vitro with desired genomic materials by reacting the viruses with transfer vectors having the genomic materials flanked with loxP sites, in the presence of cre recombination enzyme. In that method, the time-period for producing recombinant viruses is shortened. However, the production efficiency of desired recombinant viruses is still low (0.2% to 49%), thereby it is not suitable to be used as a high-throughput system for the preparation of recombinant viruses.
In addition, Luckow et al. reported a method of inserting a desired genomic cassette into a baculoviral genome by site-specific recombination in a cell. See Luckow, V. A., et al., Efficient Generation of Infectious Recombinant Baculoviruses by Site-Specific Transposon-Mediated Insertion of Foreign Genes into a Baculovirus Genome Propagated in Escherichia coli, J. Virol., August 1993, pp. 4566-4579, Vol. 67, No. 8. According to Luckow's method, a shuttle vector (sized about 130 kb) is prepared by inserting a plasmid replication origin, which replicates in bacteria, including kanamycin resistance gene and attTn7 recombination sequences, into baculoviral genome. The shuttle is named “Bacmid.” DH10Bac was generated by transforming E. coli with Bacmid and a helper plasmid, which in turn was transformed using a plasmid containing desired genes and polyhedrin promoter to prepare recombinant Bacmid in E coli. The resulting recombinant Bacmid is isolated and transferred to insect cells to obtain recombinant viruses. According to that method, baculoviruses can be obtained with relatively high efficiency by using site-specific recombination of transposon. That method, however, required complex procedures, for example, cloning desired genomic materials to Tn7 Bacmid carrier vectors; transforming DH10Bac for the recombination of Tn7 baculoviruses; selecting and isolating bacteria having Bacmid that contains the desired genomic material; purifying Bacmid DNA (sized about 130 kb) from the isolated bacteria; and transferring the DNA again to insect cells to produce recombinant virues.
Thus, non of the above-mentioned methods of the prior art have provided suitable ways to overcome the problems of a relatively long time-period in preparing recombinant viruses (about 3 to 6 weeks) and low production efficiency. Therefore, there has been a demand for the development of an improved method.
In this regard, we have carried out studies to produce recombinant viruses having a desired genomic material more rapidly and efficiently. In one embodiment, we prepared recombinant viral vectors in vitro using site-specific recombination and employing the vectors to produce recombinant viruses. Since the viral vectors obtained in vitro according to this invention can be applied to animal cells directly without further procedures like selection, the present invention not only simplifies the total procedure significantly but also provides desired recombinant viruses with up to 100% production efficiency. Thus, higher virus titer can be obtained according to the present invention.